Normal human hepatocytes are extremely difficult to obtain due to their rapid decrease in viability following autopsy. Additionally, the human liver is one of the few organs in adults capable of regeneration. However, replicative cultures of adult human hepatocytes have never been adequately established. These cells have a very limited lifespan when put into cell culture.
There are several examples of animal liver cell cultures derived from experimental laboratory animals such as rats (Tsao et. al., Exp. Cell Res., 154:38-52 (1984); Enat et al., "Proc. Nat Acad. Sci USA", 87: 1411-1415 (1984)). Rat liver epithelial cells from adult rat liver tissue have been established using serum free medium (Chessebeuf and Padieu, In Vitro, 20, 780-795 (1984); Enat et al, Proc. Natl. Acad. Sci., 81, 1411-1415 (1984)). Rat liver cells have been transformed by transfection with SV40 DNA (Woodworth et al, Cancer Res., 46: 4018-4026 (1987); Ledley et al., Proc. Nat. Acad. Sci. USA, 84: 5335-5339 (1987) but those cells are not suitable for human drug metabolism or carcinogenesis studies because of xenobiotic metabolism differences between rat and human liver cells.
Clonally-derived cultures of human hepatocytes have been reported (Kaighn and Prince, Proc. Nat. Acad. Sci., 68, 2396-2400 (1971)), but no new data has been generated to support or refute these observations. In addition, the medium used contained 17% serum. Several studies have shown that serum (Hashi and Carr J. Cell Physiol., 25, 82-90 (1985)), and more specifically transforming growth factor-beta (TGF-.beta.) present in serum (Nakaruma et al., Biochem. Biophys. Res. Commu., 133, 1042-50 (1985); Lin et al., Biochem. Biophys. Res. Commu., 143, 26-30 (1987); and Strain et al., Biochem. Biophys. Res. Commu., 145, 436-442 (1987)) cause a marked decrease in DNA synthesis of rat hepatocytes in culture. Serum was also found to cause undesired cellular differentiation and retarded replication.
Long-Term cultures of human fetal liver have been established (Salas-Prato, M. et al, In Vitro Cell Dev. Biol., 24:230-238 (1988); Sells, M. A. et al, In Vitro Cell Dev. Biol., 21:216-220 (1985)), however the inherent differences between fetal and adult liver, especially in the area of xenobiotic metabolism make adult hepatocytes a more suitable model for carcinogenesis and toxicity studies.
Rat liver epithelial cells from adult rat liver tissue have been established using serum-free medium (Chessebeuf and Padieu, In Vitro, 20, 780-795 (1984); Enat et al., Proc. Natl. Acad. Sci., 81, 1411-1415 (1984)).
Human hepatoma cell lines have been cultured and are available (e.g. Knowles et al., U.S. Pat. No. 4,393,133, Jul. 12, 1983, Human Hepatoma Derived Cell Line) but, are not usable in carcinogenesis studies because they are tumorigenic. They were also cultured in medium containing serum.
The medium described herein contains chemically denatured serum (Van Zoelen, E. J. J., J. Cell Physiol., 123:151-160 (1985)) that contains no active TGF-.beta. which we concluded inhibited DNA manufacture and cell division. This medium the uses cholera toxin, bovine pituitary extract and chemically denatured serum.
Prior cell lines have serious limitations because they are not of human origin, or are not more closely representative of normal human liver cell.